Project Summary Awkward body positions lead to an arm or leg ?falling asleep.? Upon repositioning, blood rushes back into the limb, with nerves spontaneously regaining function after being deprived of oxygen and electrolytes. While asleep, cellular and molecular processes within the ischemic limb may produce bioactive restorative and regenerative compounds that are released intravenously upon reperfusion. The method of intentional, intermittent restriction of blood flow to a limb is called Remote Ischemic Conditioning (RIC), which can be achieved by simple and cost- effective application of a tourniquet to a limb for several cycles of pre-determined duration. RIC can protect the brain and other organs from ischemic, surgical, and traumatic events, whether administered prior to or following the event. In fact, our team has demonstrated that RIC reduced biomarkers of acute damage in traumatic brain injury (TBI) patients. In pre-clinical cardiac arrest and cerebral ischemia, RIC preserves histopathology and improves functional outcome, using either pre-injury or post-injury RIC. To date, the mechanisms underlying RIC efficacy are unknown, with a dozen conflicting mechanisms proposed. TBI and other acquired neurological injuries share secondary injury processes, including inflammation and oxidative stress, which have been repeated targets of neuroprotective strategies. We propose a class of endogenous lipids derived from fatty acids, called Specialized Pro-Resolving Mediators (SPMs), as the molecular mechanism for RIC efficacy. SPMs, including the molecular families of resolvins, lipoxins, and protectins, are released from lipid bilayers after ischemia, actively resolve inflammation, and are neuroprotective in diffuse TBI, with the assumed biostability to travel in blood to the brain and the lipid structure for blood-brain barrier permeability. In this proposal, we hypothesize that RIC preserves neurological function following experimental diffuse TBI by producing SPMs, which mitigate injury-induced inflammation. To test the hypothesis, we apply RIC sequences to the hind limb of adult mice, first before and then after diffuse TBI induced by midline fluid percussion injury. Aim 1 will evaluate the efficacy of RIC sequences on preserving neurological, cognitive, and affective function over a 21 day time course post-injury. Aim 2, using the most effective sequence of RIC identified in Aim 1, will demonstrate that RIC attenuates microglial activation as an index of inflammation and produces SPMs as measured in plasma by liquid chromatography-coupled tandem mass spectrometry and commercial ELISA. Results from these aims will identify the RIC sequence necessary to improve neurological outcome from diffuse TBI. Further, we explore the production of SPMs as the molecular mechanism that targets microglial activation. Direct evidence is necessary to support the efficacy of RIC as a therapeutic approach to treat the estimated 1.7 million TBIs that occur in the United States. Ultimately, RIC could serve as a cost-effective and feasible therapy for delivering endogenous restorative and reparative compounds, such as SPMs, to improve outcome from TBI.